Composite primers comprising a 3′-DNA portion and an RNA portion are employed in previously-described DNA and RNA amplification methods, for example, as described in U.S. Pat. Nos. 6,251,639, 6,692,918, 6,815,164, 6,858,413, and 6,686,156, and in U.S. Application Nos. 2003-0087251, 2003-0003441, 2005-0014192, 2002-0164628, 2003-0215926, 2004-0023271, 2004-0005614, and 2005-0019793. Amplification of an RNA target, for example, as described in U.S. Application Nos. 20030087251, 2005-0003441, and 2005-0014192, and in PCT Application No. WO 02/072772, is initiated by a procedure for the generation of cDNA which utilizes composite primers (comprising an RNA and a 3′-DNA portion) for first strand cDNA synthesis. The RNA portion of the composite primer may comprise a sequence which does not hybridize to the target RNA sequence. The 3′-DNA portion comprises a sequence that hybridizes to the target RNA sequence. The 3′-DNA portion of the first strand cDNA primer may comprise a sequence that is complementary to the poly-A tail of mRNA, or a random sequence that is hybridizable to sequences across the RNA target sequence. Alternatively the 3′-DNA portion may comprise a sequence that is complementary to specific sequence(s) of the RNA target. The first strand cDNA synthesis is carried out by a reverse transcriptase, which extends the hybridized primer along the target RNA to form a cDNA/RNA heteroduplex. Any combination of first strand composite cDNA primers is possible. Thus, first strand cDNA synthesis may be carried out using a single composite primer, a mixture of composite primers with a random 3′.-DNA sequence, such as a random hexamer, a combination of composite primers comprising random and sequence-specific 3′-DNA portions, etc. Second strand cDNA synthesis along the first strand cDNA, and reverse transcription of the RNA portion of the first strand primer extension product, results in the formation of unique double stranded cDNA molecules with a DNA/RNA heteroduplex at one end. The heteroduplex at the end of the double-stranded cDNA is a substrate for RNase H, which can degrade RNA of this heteroduplex to generate a unique partial duplex cDNA with a single-stranded DNA portion at the 3′-end of the second strand cDNA. This single-stranded sequence comprises a sequence that is complementary to the RNA portion of the first strand cDNA composite primer utilized, and serves as a priming site for subsequent amplification using a composite DNA/RNA amplification primer. Amplification is carried out using a composite amplification primer comprising a 3′-DNA portion and an RNA portion, a DNA polymerase with strand displacement activity, and an enzyme capable of degrading RNA in an RNA/DNA heteroduplex, such as RNase H.
In one procedure for amplifying an RNA target as described above, the first strand cDNA chimeric primer comprises a RNA portion that is not hybridizable to the target RNA sequence and comprises a sequence of the chimeric amplification primer. The double stranded cDNA generated at the completion of second strand cDNA synthesis, comprises a unique DNA/RNA heteroduplex at one end. The appended sequence at one end of the double stranded cDNA comprises the RNA portion of the first strand cDNA chimeric primer and its DNA complement. Incubation of this product with an enzyme that degrades RNA in an RNA/DNA heteroduplex, such as RNase H, results in the degradation of the RNA portion of the heteroduplex, releasing a site for primer hybridization to permit amplification with the chimeric amplification primer. Insofar as the amplification priming site contains sequences that are complementary to sequences in both the first strand cDNA chimeric primer and the chimeric amplification primer, any remaining first strand cDNA chimeric primer, which was not engaged in the synthesis of first strand cDNA, is capable of competing with the amplification chimeric primer for binding at the amplification primer binding site. This competition has the potential of impacting amplification efficiency.
This competition is dependent on the concentrations of the two primers.
Whereas the chimeric amplification primer is typically added to the amplification reaction mixture at high concentration sufficient for effective productive hybridization and subsequent amplification to generate multiple copies of the single-stranded amplification products, the first strand primer is carried over into the amplification reaction mixture with the cDNA reaction mixture. The amount of first strand cDNA chimeric primer carried over into the amplification reaction mixture will be dependent on the amount added to the first strand synthesis reaction mixture, and the complexity of the primer composition. For example, the total amount of first strand chimeric primer may be particularly high when the primer employed is designed for random priming throughout the length of RNA transcripts, in contrast to a primer employed for cDNA synthesis which is initiated at defined sequences of RNA transcripts in the sample. The initiation of cDNA synthesis at specific sequences of RNA transcripts may entail hybridization and initiation of synthesis at the poly-A tail of eukaryotic mRNAs, or at sequences specific for defined internal mRNA species, such as a sequence common to a family of transcripts.
The amount (concentration) of the first strand chimeric primer added to a reaction mixture is often determined based on efficient priming and may therefore be in excess to the amount of RNA transcripts in the samples. Chimeric DNA/RNA primers designed to randomly prime cDNA synthesis throughout the length of an RNA transcript comprise a large population of primer sequences to accommodate the representation of random sequence at the 3′ end, and thus require a large concentration of the total population of primers, for effective representation of each of the priming sequences. Similarly, the total concentration of primers added for priming at multiple transcript sequences will also be higher than that required for any single primer. Effective linear amplification of the entire population of transcripts in the mixture can be achieved with the use of a single amplification primer, when all the chimeric primers employed for first strand cDNA synthesis comprise an RNA portion of the same sequence. Thus, the effective combined concentration of the RNA portion of the chimeric primer is particularly high whereas the concentration of any of the 3′ DNA portions is relatively low. The first strand cDNA chimeric primer remaining in the reaction mixture following the second strand synthesis reaction, and carried over into the amplification reaction mixture, is high when employing a random priming strategy. Insofar as all the RNA portions of the first strand chimeric primers include the RNA sequence of the chimeric amplification primer, the amplification efficiency may be impacted by competition of both sets of primers for binding to the priming site on the amplification target.
There is a need for an improved amplification procedure in which single-stranded RNA of a first single-stranded-RNA-containing primer is degraded prior to a second reaction employing a second single-stranded-RNA-containing primer, to prevent or reduce competition between the two primers for binding to the target in an amplification reaction employing the second primer.